In vitro and in vivo characterization of retinoid synthesis from β-carotene

Abstract

Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like β-carotene, through the actions β-carotene 15,15′ monooxygenase (BCMO1). As the substrates for retinoid metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact β-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms. Keywordsβ-carotene; cleavage; β-carotene 15; 15′ monooxygenase; retinoid; metabolism; cellular retinol binding protein Retinoids (vitamin A, its natural metabolites and synthetic analogs) are required for survival of mammals and are needed to support vision, normal cell differentiation and proliferation, embryonic development, normal immune function and reproduction [1]. Aside from in vision, all of the essential actions of retinoids within the body are thought to involve the transcriptional activity of retinoic acid [1]. Retinoic acid is a potent transcriptional regulator whose effects are mediated by two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) [2][3][4]. When retinoic acid is not available, RAR/RXR heterodimers bind to retinoic acid response elements in promoter of genes and form complexes with transcriptional corepressors resulting in chromatin condensation and transcriptional silencing. In the presence of retinoic acid, corepressors are released from the RAR/RXR heterodimer which is now free to recruit and bind coactivators resulting in chromatin *Corresponding author: Jisun Paik, Department of Comparative Medicine, Raitt Hall, 324, Seattle, WA 98195. Phone: 206-221-2682; Fax: 206-685-1696; E-mail: jpaik@u.washington.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript decondensation and transcriptional activation. In excess of 500 genes may be responsive to retinoic acid [5].Since ani...

β-Carotene conversion products and their effects on adipose tissue

“…Fierce et al [18] argued that BCMO1 is not essential for maintaining vitamin A stores due to their observation that liver vitamin A stores were not different between BCMO1 -/-and WT animals. However, it is likely that this discrepancy is caused by high hepatic retinoid concentrations at the beginning of the study by [18] since (a) WT and BCMO1 -/-breeders were not subjected to a diet depleted of vitamin A, and (b) the duration of the b-carotene/low vitamin A feeding period was shorter than in the study from [29]. Indeed, it has been shown that it is very difficult to trigger vitamin A deficiency in mice after weaning [53].…”

Cardiac dysfunction in β-carotene-15,15′-dioxygenase-deficient mice is associated with altered retinoid and lipid metabolism

“…For all of our experiments, we used male WT C57BL/6J mice (Jackson Laboratory) and Bco1 Ϫ/Ϫ mice congenic in the C57BL/6J genetic background. Bco1 Ϫ/Ϫ mice, which were originally described for the mixed C57BL/6;129svJ genetic background (14), have undergone 10 backcrosses with C57BL/6J mice to render them congenic in this inbred background. Genotypes of Bco1 Ϫ/Ϫ mice were determined by PCR using a previously described protocol (14).…”

Inhibition of Retinoic Acid Biosynthesis by the Bisdichloroacetyldiamine WIN 18,446 Markedly Suppresses Spermatogenesis and Alters Retinoid Metabolism in Mice

“…An open reading frame was subcloned into a pET28 bacterial expression vector containing a 3Ј histidine tag, and the resulting sequence was verified through DNA sequencing (University of Washington DNA sequencing facility). Expression of the enzyme was induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside, and purification was performed using His⅐Bind resin (Novagen, San Diego, CA) as described previously (16). Purified enzyme was dialyzed against 20 mM Hepes buffer (pH 8.5) containing 150 mM KCl and 1 mM EDTA.…”

“…Retinol and Retinyl Ester Analysis-Retinoid extractions and detections were carried out as described previously (16,22 Retinoic Acid Analysis from Testes-Intratesticular retinoic acid (RA) concentrations were measured by LC-MS/MS as described previously with a few modifications to the sample preparation (23). All sample processing, preparation, and extraction were conducted on ice under red light to minimize degradation of retinoic acids.…”